1. Field of the Invention
The invention relates to a process for purifying L-cysteine from an L-cysteine-containing fermenter broth.
2. Background Art
L-Cysteine is an amino acid which, owing to good solubility in water and high sensitivity of the SH group toward a multiplicity of reagents, for example toward oxidizing agents, can be purified and isolated only with very great difficulties and great expense. In contrast, the amino acid L-cystine can be purified and isolated readily and with very high purity, even from complex substance mixtures such as, for example, protein hydrolysates or L-cystine-containing fermenter broths, owing to very low solubility in water and comparatively high stability, for example toward oxidizing agents. DE10040176A1, for example, describes a very simple process for isolating L-cystine from cell-containing suspensions or fermenter broths.
Therefore, L-cysteine is industrially produced from complex mixtures such as, protein hydrolysates (e.g. from human hair or animal sources (e.g. feathers or bristles)) or appropriate fermenter broths by firstly isolating the sparingly soluble L-cystine in a purified form. L-Cysteine or other L-cysteine derivatives which may be present in such complex substance mixtures are specifically and as completely as possible converted to L-cystine, for example by way of oxidation. L-cystine is then reduced by way of subsequent reduction (e.g. by electrolysis) to give L-cysteine. However, this process has disadvantages, since L-cysteine must be prepared in a complicated, two-stage process via the intermediate L-cystine.
EP0250987B1 describes direct isolation of L-cysteine from a solution containing L-cysteine, L-cystine, L-serine and an inorganic salt. Firstly, L-cystine and the inorganic salt are crystallized at at least 20° C. by adding hydrogen chloride and removed by filtration. From the remaining solution which still contains L-cysteine and L-serine, L-cysteine hydrochloride monohydrate is then crystallized and isolated with high purity at no more than 10° C. However, the process is limited to solutions which contain L-cysteine, L-cystine, L-serine and an inorganic salt and does not enable L-cysteine to be obtained in high yield and purity from complex substance mixtures such as protein hydrolysates or fermentation broths.
EP1645623A1, EP1298200B1, US20050221453A1, EP1234874A1 and EP1571223A2 describe isolating L-cysteine from fermentation broths by means of a combination of ion exchange, crystallization and other known methods. However, no information on the specific procedure, or on the yields and purities obtained, is given.
EP 1650296A1 describes isolating L-cysteine from fermentation broths by removing the solids by centrifugation or membrane filtration and subsequently isolating and purifying the amino acid by means of ion exchange, concentration and crystallization. Here too, no information is given on the specific procedure, or on the yields and purities obtained.
Thus, the problem of directly and cost-effectively purifying and producing L-cysteine from complex substance mixtures such as, for example, L-cysteine-containing fermenter broths of a microorganism remains unsolved. No process has been disclosed which can be implemented on an industrial scale and by which L-cysteine can be obtained in high purity and/or yield from L-cysteine-containing fermenter broths in a cost-effective, direct manner and without derivatization, for example by means of oxidation to give L-cystine and subsequent reduction to give L-cysteine.
EP0885962B1, EP0858510B1 and EP1220940B1 describe processes for fermentative production of L-cysteine. These processes enable fermentation broths containing large amounts of L-cysteine to be accessed in a cost-effective manner.
Such an L-cysteine-containing fermenter broth is an extremely complex substance mixture. Apart from L-cysteine it usually contains L-cystine which is readily formed from L-cysteine under the fermentation conditions, in particular due to oxidation by available oxygen. Furthermore, in the presence of aldehydes or ketones, corresponding hemithioketals and/or thiazolidine derivatives of L-cysteine may be present, as described, for example, in EP0885962B1. The fermenter broths may also contain small amounts of other amino acids or derivatives thereof. They also usually contain carbohydrates, salts of organic and inorganic cations and anions, for example alkali metal salts and alkaline earth metal salts, and traces of heavy metal salts (e.g. Fe, Cu, Mn, Zn, etc.), dyes and further contaminations and additives such as, for example, undesired metabolic products of the microorganisms used in the fermentation. The fermenter broths, as described, for example, in EP0885962B1, EP0858510B1 and EP1220940B1, may further also contain the raw materials and ingredients used in the fermentation, for example customary carbon sources such as glucose, lactose, starch and the like, nitrogen sources such as ammonia/ammonium or proteins or protein hydrolysates and the like, and sulfur sources such as, for example, sulfide, sulfite, sulfate, thiosulfate or dithionite and the like. Since L-cysteine is a sulfur-containing amino acid, a sulfur source such as, for example, sulfide, sulfite, sulfate, thiosulfate or dithionite is usually fed in during the fermentation, in order to provide a sufficient amount of sulfur required for the formation of L-cysteine. The fermenter broths furthermore also contain dissolved oxygen, due to the oxygen introduced during fermentation. Said fermenter broths usually have a pH of 7, as described, for example, in EP0885962B1.
L-cysteine can be oxidized, for example to L-cystine, in a fermenter broth or another solution by any oxidizing agents capable of oxidizing SH groups. Apart from L-cystine as the primary product of the oxidation of L-cysteine, more highly oxidized compounds of L-cysteine and/or L-cystine may also be produced. The result of the presence of such oxidizing agents in L-cysteine-containing fermenter broths or L-cysteine-containing solutions is therefore an immediate reduction in the yield of L-cysteine.
Examples of oxidizing agents capable of oxidizing SH groups (and therefore also L-cysteine) at pH<5 are oxygen and sulfur-oxygen compounds. These oxidizing agents are normally present in variable amounts in L-cysteine-containing fermenter broths. Sulfur-oxygen compounds such as, for example, thiosulfate either are added directly as a sulfur source during fermentation, as described in EP0885962B1, for example, or may be produced from other added sulfur sources such as, for example, sulfide, sulfite, sulfate, thiosulfate or dithionite and the like during fermentation. Thus it is possible, for example, for sulfide or hydrogensulfide, inter alia, to be readily oxidized to thiosulfate by oxygen introduced during the fermentation. The complex chemistry of sulfur-oxygen compounds and formation thereof has also been described in detail, for example, in Hollemann-Wiberg, Lehrbuch der Anorganischen Chemie, 91st to 100th edition, Walter de Gruyter, Berlin-New York, 1985, pp. 485-523.
L-Cysteine is oxidized particularly readily by oxygen as an oxidizing agent, preferably at high pH. An L-cysteine solution can be stabilized against oxidation by oxygen by lowering the pH of the solution. For example, aqueous solutions of L-cysteine hydrochloride, preferably in hydrochloric acid, are known to be substantially more stable against oxidation by oxygen than, for example, aqueous solutions of L-cysteine with a pH of 7 or higher.
In contrast, the oxidizing power toward SH groups, and thus also toward L-cysteine, of a multiplicity of sulfur-oxygen compounds increases with decreasing pH, in some cases markedly. These oxidizing agents may be present in L-cysteine-containing fermenter broths or else L-cysteine-containing solutions purified therefrom and oxidize L-cysteine only to a very low extent, if at all, at pH values occurring during fermentation, for example, but oxidize L-cysteine in some cases very well with decreasing pH, particularly at pH<5.
Examples of sulfur-oxygen compounds capable of oxidizing SH groups and therefore also L-cysteine are sulfur dioxide or sulfur trioxide, for example. Sulfur dioxide is also released upon acidification of sulfite solutions and is then able to oxidize SH groups. Thiosulfate which is a preferred sulfur source used in the fermentation of L-cysteine and which practically does not oxidize L-cysteine at pH>5, preferably at a pH of 7, as present during fermentation, oxidizes L-cysteine at a pH of <5, with oxidizing power and rate of oxidation increasing with decreasing pH.
A number of other sulfur-oxygen compounds and their ability to oxidize SH groups, preferably in acidic medium, are also described in Hollemann-Wiberg, Lehrbuch der Anorganischen Chemie, 91st-100th edition, Walter de Gruyter, Berlin-New York, 1985, pp. 485-523.
There are furthermore a number of compounds which can catalyze oxidation of SH groups. Thus, for example, heavy metal salts are known to be able to effectively catalyze oxidation of cysteine to cystine. Heavy metal salts such as iron salts or zinc salts are also frequently essential additives in fermentations and are therefore also present in the corresponding fermentation broths, usually in small amounts.
If an L-cysteine-containing solution or fermenter broth contains an oxidizing agent capable of oxidizing L-cysteine to, for example, L-cystine at pH<5, then this results in an immediate loss of yield, if such solutions or fermenter broths are acidified to pH<5. Since the preferred final product, L-cysteine hydrochloride monohydrate, is crystallized from strongly acidic solutions, a loss of yield of up to 100% of the L-cysteine present is possible, depending on the oxidizing agent content with respect to L-cysteine.